{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,14]],"date-time":"2026-10-14T18:50:26Z","timestamp":1792003826826,"version":"4.4.0"},"reference-count":41,"publisher":"American Chemical Society (ACS)","issue":"40","license":[{"start":{"date-parts":[[2026,8,13]],"date-time":"2026-08-13T00:00:00Z","timestamp":1786579200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["19H05685"],"award-info":[{"award-number":["19H05685"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["22H02192"],"award-info":[{"award-number":["22H02192"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["22H05130"],"award-info":[{"award-number":["22H05130"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["22KJ1046"],"award-info":[{"award-number":["22KJ1046"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["25K01955"],"award-info":[{"award-number":["25K01955"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["JPJSA3F20250002"],"award-info":[{"award-number":["JPJSA3F20250002"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,10,14]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>The diazo group is a highly valuable functional group in organic synthesis; however, its conventional preparation requires harsh conditions. Here, we provide a systematic analysis of ATP-dependent diazotases from actinomycetes that catalyze the condensation of nitrite with aromatic amines. Sequence similarity network analysis of AMP-binding enzymes revealed two families (type Ia\/Ib), with type Ib further classified into 14 groups. In vitro assays confirmed the diazotization activities of the diazotases from 7 groups in the type Ib family, each displaying distinct substrate preferences. Remarkably, diazotases in group 3 exhibited broad substrate specificity and superior catalytic efficiency. The dimeric structure of a representative enzyme, Mco01_40450, was determined at 3.08 \u00c5 resolution by cryogenic electron microscopy with C2 symmetry. Subsequent symmetry expansion and 3D classification revealed the structure with AMP, pyrophosphate, and 4-aminohydrocinnamic acid bound to the active site at a resolution of 3.16 \u00c5. Expansion of the substrate-binding pocket and its entrance via amino acid substitution enabled the reaction of bulkier aromatic amines. Unlike classical chemical diazotization, Mco01_40450 operates under mild aqueous conditions and is compatible with sensitive protecting groups. Our findings highlight the structural and functional diversity of diazotases and establish group 3 diazotases as promising, engineerable biocatalysts for selective and efficient diazo installation.<\/jats:p>","DOI":"10.1021\/jacs.6c11480","type":"journal-article","created":{"date-parts":[[2026,8,13]],"date-time":"2026-08-13T21:02:21Z","timestamp":1786654941000},"page":"43239-43248","source":"Crossref","is-referenced-by-count":0,"title":["Promiscuous ATP-Dependent\nDiazotases Discovered by\nComprehensive Genome Mining Based on Sequence Similarity Network Analysis"],"prefix":"10.1021","volume":"148","author":[{"given":"Jiayu","family":"Ning","sequence":"first","affiliation":[{"name":"The University of Tokyo , , 1-1-1 Yayoi , Bunkyo-ku, ,","place":["Tokyo, Japan, 113-8657"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Seiji","family":"Kawai","sequence":"additional","affiliation":[{"name":"The University of Tokyo , , 1-1-1 Yayoi , Bunkyo-ku, ,","place":["Tokyo, Japan, 113-8657"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4905-5320","authenticated-orcid":true,"given":"Yohei","family":"Katsuyama","sequence":"additional","affiliation":[{"name":"The University of Tokyo , , 1-1-1 Yayoi , Bunkyo-ku, ,","place":["Tokyo, Japan, 113-8657"]},{"name":"The University of Tokyo , , Bunkyo-ku, ,","place":["Tokyo, Japan, 113-8657"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yasuo","family":"Ohnishi","sequence":"additional","affiliation":[{"name":"The University of Tokyo , , 1-1-1 Yayoi , Bunkyo-ku, ,","place":["Tokyo, Japan, 113-8657"]},{"name":"The University of Tokyo , , Bunkyo-ku, ,","place":["Tokyo, Japan, 113-8657"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,13]]},"reference":[{"issue":"4","key":"2026101414081055100_cit1","doi-asserted-by":"publisher","first-page":"1091","DOI":"10.1021\/cr00028a010","article-title":"Organic Synthesis with \u03b1-Diazo Carbonyl Compounds","volume":"94","author":"Ye","year":"1994","journal-title":"Chem. Rev."},{"issue":"28","key":"2026101414081055100_cit2","doi-asserted-by":"publisher","first-page":"6577","DOI":"10.1016\/j.tet.2008.04.074","article-title":"Recent Studies on the Reactions of \u03b1-Diazocarbonyl Compounds","volume":"64","author":"Zhang","year":"2008","journal-title":"Tetrahedron"},{"issue":"18","key":"2026101414081055100_cit3","doi-asserted-by":"publisher","first-page":"1797","DOI":"10.1002\/anie.199417971","article-title":"Ligand Effects on the Chemoselectivity\nof Transition\nMetal Catalyzed Reactions of \u03b1-Diazo Carbonyl Compounds","volume":"33","author":"Padwa","year":"1994","journal-title":"Angew. Chem., Int. Ed. Engl."},{"issue":"25","key":"2026101414081055100_cit4","doi-asserted-by":"publisher","first-page":"9253","DOI":"10.1021\/jo061411m","article-title":"Perspective\non Dirhodium Carboxamidates as Catalysts","volume":"71","author":"Doyle","year":"2006","journal-title":"J.\nOrg. Chem."},{"issue":"1","key":"2026101414081055100_cit5","doi-asserted-by":"publisher","first-page":"125","DOI":"10.1016\/S1381-1169(02)00201-7","article-title":"Catalytic\nAsymmetric C-H Activation of Sp3 Hybridized C-H Bonds by Means of\nCarbenoid C-H Insertions: Applications in Organic Synthesis","volume":"189","author":"Davies","year":"2002","journal-title":"J. Mol. Catal. A:Chem."},{"issue":"1","key":"2026101414081055100_cit6","doi-asserted-by":"publisher","first-page":"50","DOI":"10.1039\/b000708k","article-title":"Catalytic Enantioselective\nRearrangements\nand Cycloadditions Involving Ylides from Diazo Compounds","volume":"30","author":"Hodgson","year":"2001","journal-title":"Chem. Soc. 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Chem."},{"issue":"2","key":"2026101414081055100_cit21","doi-asserted-by":"publisher","first-page":"73","DOI":"10.1038\/nchembio.1991","article-title":"A Nitrous Acid Biosynthetic\nPathway\nfor Diazo Group Formation in Bacteria","volume":"12","author":"Sugai","year":"2016","journal-title":"Nat.\nChem. Biol."},{"issue":"11","key":"2026101414081055100_cit22","doi-asserted-by":"publisher","first-page":"911","DOI":"10.1038\/s41429-018-0088-1","article-title":"Novel Desferrioxamine\nDerivatives Synthesized Using the Secondary Metabolism-Specific Nitrous\nAcid Biosynthetic Pathway in Streptomyces Davawensis","volume":"71","author":"Hagihara","year":"2018","journal-title":"J. Antibiot."},{"issue":"45","key":"2026101414081055100_cit23","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202211728","article-title":"Bacterial Avenalumic Acid Biosynthesis Includes Substitution\nof an Aromatic Amino Group for Hydride by Nitrous Acid Dependent Diazotization","volume":"61","author":"Kawai","year":"2022","journal-title":"Angew. Chem. Int. Ed."},{"key":"2026101414081055100_cit24","doi-asserted-by":"publisher","first-page":"1","DOI":"10.3762\/bjoc.20.1","article-title":"Identification of the p-Coumaric Acid Biosynthetic Gene Cluster in Kutzneria Albida: Insights into the Diazotization-Dependent Deamination\nPathway","volume":"20","author":"Kawai","year":"2024","journal-title":"Beilstein J. Org. Chem."},{"issue":"8","key":"2026101414081055100_cit25","doi-asserted-by":"publisher","first-page":"1821","DOI":"10.1021\/acschembio.3c00248","article-title":"Identification and Analysis of the Biosynthetic Gene\nCluster for the Hydrazide-Containing Aryl Polyene Spinamycin","volume":"18","author":"Kawai","year":"2023","journal-title":"ACS Chem. Biol."},{"issue":"12","key":"2026101414081055100_cit26","doi-asserted-by":"publisher","first-page":"1305","DOI":"10.1038\/s41589-021-00895-3","article-title":"Biosynthesis\nof Triacsin Featuring an N-Hydroxytriazene Pharmacophore","volume":"17","author":"Del Rio Flores","year":"2021","journal-title":"Nat. Chem. Biol."},{"issue":"4","key":"2026101414081055100_cit27","doi-asserted-by":"publisher","first-page":"1622","DOI":"10.1021\/jacs.1c10369","article-title":"Biosynthesis\nof Tasikamides via Pathway Coupling and Diazonium-Mediated Hydrazone\nFormation","volume":"144","author":"Ma","year":"2022","journal-title":"J. Am. Chem. Soc."},{"issue":"16","key":"2026101414081055100_cit28","doi-asserted-by":"publisher","DOI":"10.1002\/cbic.202500377","article-title":"Functional\nCharacterization of a\nDiazo-Forming Enzyme in Meroterpenoid Biosynthesis","volume":"26","author":"Candra","year":"2025","journal-title":"ChemBioChem"},{"issue":"18","key":"2026101414081055100_cit29","doi-asserted-by":"publisher","first-page":"7912","DOI":"10.1039\/D4SC08319A","article-title":"Biosynthesis of the Tetrahydroxynaphthalene-Derived\nMeroterpenoid Furaquinocin via Reductive Deamination and Intramolecular\nHydroalkoxylation of an Alkene","volume":"16","author":"Noguchi","year":"2025","journal-title":"Chem. Sci."},{"issue":"27","key":"2026101414081055100_cit30","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202505851","article-title":"Structural Basis for\nthe Catalytic Mechanism of ATP-Dependent\nDiazotase CmaA6","volume":"64","author":"Kawai","year":"2025","journal-title":"Angew. Chem., Int. Ed."},{"issue":"18","key":"2026101414081055100_cit31","doi-asserted-by":"publisher","first-page":"10319","DOI":"10.1002\/anie.202100462","article-title":"Complete Biosynthetic\nPathway of Alazopeptin, a Tripeptide\nConsisting of Two Molecules of 6-Diazo-5-oxo-l-norleucine and One\nMolecule of Alanine","volume":"60","author":"Kawai","year":"2021","journal-title":"Angew. Chem., Int. Ed."},{"issue":"12","key":"2026101414081055100_cit32","doi-asserted-by":"publisher","first-page":"2066","DOI":"10.1038\/s41557-024-01646-2","article-title":"Enzymatic Synthesis of Azide by a\nPromiscuous N-Nitrosylase","volume":"16","author":"Del Rio Flores","year":"2024","journal-title":"Nat. Chem."},{"issue":"10","key":"2026101414081055100_cit33","doi-asserted-by":"publisher","first-page":"811","DOI":"10.1021\/cb900156h","article-title":"Conformational\nDynamics in the Acyl-CoA Synthetases, Adenylation Domains of Non-Ribosomal\nPeptide Synthetases, and Firefly Luciferase","volume":"4","author":"Gulick","year":"2009","journal-title":"ACS Chem. Biol."},{"issue":"1","key":"2026101414081055100_cit34","first-page":"205","article-title":"A New Generation\nof Homology Search Tools Based on Probabilistic Inference","volume":"23","author":"Eddy","year":"2009","journal-title":"Genome Inf."},{"issue":"5","key":"2026101414081055100_cit35","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pcbi.1000069","article-title":"A Probabilistic\nModel of Local Sequence Alignment That Simplifies Statistical Significance\nEstimation","volume":"4","author":"Eddy","year":"2008","journal-title":"PLoS Comput. Biol."},{"issue":"10","key":"2026101414081055100_cit36","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pcbi.1002195","article-title":"Accelerated\nProfile HMM Searches","volume":"7","author":"Eddy","year":"2011","journal-title":"PLoS Comput. Biol."},{"issue":"D1","key":"2026101414081055100_cit37","doi-asserted-by":"publisher","first-page":"D412","DOI":"10.1093\/nar\/gkaa913","article-title":"Pfam: The Protein Families\nDatabase in 2021","volume":"49","author":"Mistry","year":"2021","journal-title":"Nucleic Acids Res."},{"issue":"14","key":"2026101414081055100_cit38","doi-asserted-by":"publisher","first-page":"168018","DOI":"10.1016\/j.jmb.2023.168018","article-title":"EFI-EST,\nEFI-GNT, and EFI-CGFP: Enzyme\nFunction Initiative (EFI) Web Resource for Genomic Enzymology Tools","volume":"435","author":"Oberg","year":"2023","journal-title":"J. Mol. 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Chem."},{"key":"2026101414081055100_cit41","doi-asserted-by":"publisher","DOI":"10.1002\/cbic.202400687","article-title":"Production of Phenyldiazene\nDerivatives Using the Biosynthetic Pathway of an Aromatic Diazo Group-Containing\nNatural Product from an Actinomycete","volume":"26","author":"Kawai","year":"2025","journal-title":"ChemBioChem"}],"container-title":["Journal of the American\nChemical Society"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/jacs.6c11480","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jacsat\/article-pdf\/148\/40\/43239\/66537909\/jacs.6c11480.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jacsat\/article-pdf\/148\/40\/43239\/66537909\/jacs.6c11480.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,14]],"date-time":"2026-10-14T18:29:14Z","timestamp":1792002554000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jacsat\/article\/148\/40\/43239\/5254957\/Promiscuous-ATP-Dependent-Diazotases-Discovered-by"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,13]]},"references-count":41,"journal-issue":{"issue":"40","published-online":{"date-parts":[[2026,8,13]]},"published-print":{"date-parts":[[2026,10,14]]}},"URL":"https:\/\/doi.org\/10.1021\/jacs.6c11480","relation":{},"ISSN":["0002-7863","1520-5126"],"issn-type":[{"value":"0002-7863","type":"print"},{"value":"1520-5126","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,13]]}}}